Ionizing radiation is a carcinogen that induces malignancies in human and animal model systems. It is known to have the potential to participate at many points in the multistep process of cancer development. This multistep process reflects the cellular evolution from normal through initiated, preneoplastic and premalignant to neoplastic and malignant cells. We have recently demonstrated the induction of a malignant phenotype in human cells using the concerted action of a DNA tumor virus and ionizing radiation. This transformation of immortalized human epidermal keratinocytes in vitro by ionizing radiation appeared to involve multiple events. The first was the acquisition of unlimited growth potential as a result of infection with the AD12-SV40 hybrid virus. The second involved further changes in cellular growth properties as a result of successive exposures to ionizing radiation. This radiation-induced transformation resulted in morphological alterations, growth in soft agar and the ability to form poorly differentiated squamous cell carcinomas in athymic nude mice. Subsequently, we have been able to demonstrate the presence of dominant transforming human sequences in this radiation-induced malignant phenotype using the NIH/3T3 tumorigenicity assay. These observations indicate that the use of immortalized/initiated human epidermal keratinocytes provides a model in which to study the molecular events involved in the later phases of radiation carcinogenesis. The goal of this proposal is to clone and identify the cellular genes that have been activated or inactivated during the development of this radiation-induced malignant phenotype. Once isolated and identified, we propose to verify experimentally their roles in the process of radiation-induced transformation. The results of these experiments will offer an insight into the molecular basis for radiation carcinogenesis and may provide a genetic basis for the assessment of human subjects at risk.